Tuberculosis kills over 1.5 million people each year and the emergence of multiple and extensively drug resistant strains demands new approaches for the diagnosis, prevention, and cure of TB. Construction and analysis of recombinant and mutant strains of M. tuberculosis provides fundamental genetic tools for understanding virulence mechanisms and drug resistance, but there remain numerous impediments to efficient genetic dissection of M. tuberculosis. One specific problem is that of plasmid instability, such that extrachromosomal plasmids provide suitably good expression of foreign genes, but are inherently unstable and are rapidly lost in the absence of direct selection. This is of particular concern for the use of recombinant strains in animal experiments, and is a substantial impediment to the construction of live recombinant vaccine strains. A large collection of over 1000 completely sequenced mycobacteriophages reveals a subset of temperate phages that encode partitioning cassettes (parABS) suggesting that they establish extrachromosomal autonomously replicating prophages with both an origin of replication and the segregation apparatus for prophage maintenance and stability. Identification of the functional components of the phage-encoded parABS systems and incorporation into extrachromosomal plasmids will confer substantial stability, which can then be further optimized by addition of phage-encoded addiction cassettes. Furthermore, the phage- encoded origin of replication can be exploited to develop new low-copy number stably maintained mycobacterial plasmids, and the specificity of ParB recognition of the centromere- like parS site can be exploited for detailed analysis of tuberculosis chromosomal dynamics during bacterial growth.